Biodiesel is a liquid fuel originating from renewable resources, i.e. biomass, alternative to fossil oil products, containing alkyl esters (mostly methyl and ethyl) of long chain fatty acids. They are obtained by catalytic transesterification of triacylglycerides, present in vegetable oils (i.e. olive, sunflower, etc) and/or animal fats, with a primary aliphatic alcohol (i.e. methanol, ethanol, usually) acting as acyl acceptor.
The industrial production of biodiesel in Europe, USA and Japan is carried out by chemical processes based on transesterification catalyzed by strong bases or such as for example NaOH, or KOH, which are toxic and corrosive by nature (see U.S. Pat. No. 354,878; US Pat. No 2003/0032826). Biodiesel production by this technology shows several problems, such as the difficulty to recover the by-product glycerol, the necessity to eliminate the base catalyst from the reaction medium by washing with water, which produces large amounts of alkaline wastes. Additionally, the initial content of water and free fatty acids in the vegetable oil or fat used as substrate is a serious drawback, because of the decrease in biodiesel yield due to the undesirable by-products (i.e. soap, emulsions, etc) generated, which makes it necessary to use refined vegetable oils.
Over the last years, the advantages of using immobilized enzymes onto solid supports as catalysts to synthesize products with industrial interest have been widely demonstrated. In this way, the enzymatic synthesis of biodiesel by transesterification from vegetable oils has been widely described (see U.S. Pat. No. 6,398,707 B1; Pat. WO 2006/050589 A1; Pat. WO 2007/055661 A1; Pat. EP 1 705 238 A1; Shimada, Y. et al., 1999, J. Am. Oil Chem. Soc., 76, 789-793; Shimada, Y. et al., 2002, J Mol. Catal B: Enzymatic, 17, 133-142; Soumanou M. and Bornscheuer U. T, 2003, Enzyme Microb. Technol., 33, 97-103; Mahabubur M. D. et al., 2006, Biocatal. Biotrans., 24, 257-262; Royon D. et al., 2006, Biores. Technol., 98, 648-653; Al-Zuhair et al., 2006, Biochem. Eng. J., 30, 212-217). Immobilized lipases used are for instance NOVOZYME 435 (lipase B from Candida antartica), LIPOZYME TL (lipase from Thermomyces lanuginose), LIPOZYME RM (lipase from Rhizomucor miehei).
However, the industrial interest for an enzymatic process in a non-aqueous medium will greatly rely on the activity, stability and recycling ability of the biocatalyst which is used and the easy separation of the biodiesel from reaction mixture and glycerol. In this regards, the synthesis of biodiesel by using immobilized lipases shows several disadvantages, which constitutes a handicap towards their exploitation at industrial scale. Firstly, it is necessary to point out the low solubility and/or immiscibility of both triacylglycerides (i.e. vegetable oil and/or animal fats) and primary aliphatic alcohols (i.e. methanol, ethanol, etc.) assayed in the transesterification reaction, resulting in biphasic systems. Secondly, the hydrophilic nature of these alcohols is detrimental to the immobilized lipases when introduced into the reaction mixture, catalyst particles getting impregnated with the alcohol, which seriously limits the accessibility of triacylglycerides into the enzyme microenvironment and induces the enzyme deactivation. The final result is an important loss in enzyme efficiency which limits the turnover frequency of the enzyme and the number of recycling operations. Furthermore, the transesterification reaction catalyzed by the enzyme yields both the fatty acid alkyl esters (biodiesel) and the by-product glycerol which provokes a continuous poisoning of biocatalyst until full deactivation.
Several strategies have been developed to overcome all the constraints mentioned above. However, the most popular strategy to improve the catalytic efficiency of enzymes in the synthesis of biodiesel is the solubilisation of substrates (triacylglycerides and methanol) in organic solvents having medium polarity, such as, tert-butanol, 2-propanol, 2-butanol or tetrahydrofuran, in a ratio higher than 30% v/v with respect to the substrates. These systems lead to a clear increase in the overall price of the process by both the consumption of the solvent and the necessity to purify the biodiesel product by distillation.
The use of volatile organic solvents, as reaction media in enzymatic processes, shows several disadvantages such as, the necessity of recovery, as a consequence of the high price and environment impact. The use of clean and sustainable technologies in the chemical industry, also called Green Chemistry, is one of the main developments for the near future in our society. In this way, ionic liquids (ILs) have recently emerged as new green solvents and/or reaction media for chemical processes, because of their capacity to constitute a clean alternative, non-pollutant and reusable as compared to volatile organic solvents (VOS).
The use of ILs as reaction media in enzymatic transformations is very recent: the first reference has been published in 2000 (see Erbeldinger, M. et al., 2000, Biotechnol. Prog. 16, 1129-1131; Sheldon, R. A. 2005, Green Chem., 7, 267-278). This discovery has had a great impact over the last six years, as a consequence of the excellent activity and stability displayed by the numerous assayed enzymes. Lipases are the most referenced in both free and immobilized forms (see Madeira Lau R. et al., 2000, Org Lett. 2, 4189-4191; Lozano, P., et al 2001, Biotechnol. Lett. 23, 1529-1533; Lozano, P. et al., 2001, Biotechnol. Bioeng. 75, 563-569). Today, ILs are the most interesting green alternative to volatile organic solvents for development of biocatalytic processes in non aqueous systems, including at extremely high temperatures (i.e. 150° C.) (see Lozano, P. et al., 2003, Biotechnol. Prog. 19, 380-382).
The use of some ILs for the synthesis of biodiesel by using chemical or enzymatic catalysis has recently been described. In all cases, the assayed ILs were based on short-chain 1,3-dialkylimidazolium cations (e.g. 1-ethyl-3-methylimidazolium [Emim], 1-butyl-3-methylimidazolium [Bmim], or 1-hexyl-3-methylimidazolium [Hmim]). Thus, the IL 1-butyl-3-methylimidazolium tetrachloro-indate [Bmim][InCl4] (Neto. et al., 2007, J. Catal. 249, 154-161) has been tested for the synthesis of biodiesel from vegetable oils by using a stannous complex [Sn(3-hydroxy-2-methyl-4-pyrone)2(H2O)2] as chemical catalyst. A fast complete deactivation of the system was observed after the first cycle of use. In the same way, Brönsted acidic ILs (e.g. 1-butylsulfonic-3-methylimidazolium sulphate) have been assayed as chemical catalyst to produce biodiesel by the transesterification of cottonseed oil with methanol at temperatures higher than 150° C. (Wu, Q. et al., 2007, Ind. Eng. Chem. Res., 46, 7955-7960). For the case of enzymatic catalysis, the use of ILs based on short-chain 1,3-dialkylimidazolium cation (i.e. 1-butyl-3-methylimidazolium hexafluorophosphate [Bmim][PF6] or 1-butyl-3-methylimidazolium tetrafluoroborate [Bmim][BF4]) were shown as non fully-appropriate reaction media for the synthesis of biodiesel (Shunitha, S., et al., 2007, Biotechnol. Lett., 29, 1881-1885; Ha, S. H. et al., 2007, Enzyme Microb. Technol., 41, 480-483; Gamba, M. et al., 2008, Adv. Synth. Catal., 350, 160-164). In all cases, the low solubility of triacylglycerides in the assayed ILs resulted in two-phase reaction media that provided low enzymatic activity, ending in 24 h reaction times to reach full conversion of triglycerides into FAMEs.